Method for regulating cell death

ABSTRACT

A method for regulating cell death in a plant, includes the steps of: transforming a plant cell with a polynucleotide containing a gene encoding DS9 or a homologue thereof or a part of the gene; and redifferentiating the transgenic plant cell to obtain a plant. The DS9 or the homologue thereof is an ATP-dependent Zn-type metalloprotease. The polynucleotide decreases or increases production of the ATP-dependent Zn-type metalloprotease in the plant cell, whereby cell death of a cell in the plant is promoted or suppressed.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for regulating celldeath. More specifically, the present invention relates to a method forproviding a plant to which is conferred resistance to variousenvironmental stresses by regulating an expression level of a cell deathregulatory gene.

[0003] 2. Description of the Related Art

[0004] When a plant is infected with a pathogen, e.g., virus, bacteria,filamentous fungi, and viroid, the plant shows either of the followingreactions: 1) allowing pathogen to grow by spreading through the entirebody of the plant, whereby the plant gets disease or 2) enclosingpathogen in an infected site so as to prevent if from spreading throughthe entire body of the plant, whereby the plant is provided withresistance to the pathogen. The latter reaction of a plant against thepathogen is called a hypersensitive response or reaction (HR). It isknown that, in this reaction, cell death locally occurs in an infectedsite to form necrotic lesions. Such a formation of necrotic lesionsinvolved in pathogen infection is a typical resistance reaction of aplant, which is considered as an example of programmed cell death.However, the molecular mechanism of this reaction remains unclear.

[0005] The HR does not occur in all plants. The HR is believed to occurwhen a plant intrinsically contains a gene which recognizes a product ofa pathogenic gene derived from infecting pathogen. In the case wheresuch a gene is not present, the HR does not occur, and a plant is notresistant against the pathogen infection.

[0006] The HR of tobacco against tobacco mosaic virus (TMV) infection isa model system which has been conventionally used for studying the HR ofa plant.

[0007] An N gene is one of the cell death regulatory genes involved inthe HR (i.e., cell death) due to TMV infection. It is reported thattobacco having the N gene (NN tobacco) shows the HR against TMVinfection, but tobacco having no N gene (nn tobacco) does not show theHR (Holmes, Phytopathology, 28, 553, (1938)). The HR of the NN tobaccooccurs only at 24° C or lower. It does not occur at 28° C. or higher.Therefore, it has been considered that both the N gene and thetemperature condition are required for inducing the HR in a TMV-infectedcell.

[0008] However, the inventors' group has found that, in the case wherethe NN tobacco is treated with actinomycin D (AMD) and heat (50° C., 2minutes), the HR is induced in the NN tobacco against TMV infection evenunder the temperature condition of 30° C. at which the HR does notusually occur. Furthermore, the HR was also induced against TMVinfection in the nn tobacco having no N gene, in the case where the nntobacco was similarly treated with AMD and heat. Because of this, it wasclarified that cell death against TMV infection may occur irrespectiveof the presence or absence of the N gene and the temperature condition(Shimomura and Ohashi, Virology, 43, 531, (1971); Ohashi and Shimomura,Virology, 48, 601 (1972)). It is known that AMD inhibits DNA-dependentRNA synthesis in a nucleus (Reich et al., Proceedings of the NationalAcademy of Sciences, 48, 1238 (1962)). Thus, a possibility was shownthat a novel cell regulatory gene may be present in a plant, and thatthe HR may be induced by suppression of transcription of the gene,followed by suppression of synthesis of proteins.

[0009] It is considered that if the above-mentioned cell deathregulatory gene is identified, cell death of a plant can be regulated(promoted or suppressed) by controlling an expression level of the gene.In particular, it is an important task in the agricultural field toprovide a plant which is conferred with resistance to environmentalstress by regulating cell death.

[0010] However, the cell death regulatory gene as described above hasnot been identified. To the extent that the inventors are aware, therehas been no study for providing a plant which is conferredenvironmental-stress resistance by regulating an expression level ofsuch a gene to promote or suppress cell death.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method for regulating cell deathin a plant of the present invention including the steps of: transforminga plant cell with a polynucleotide containing a gene encoding DS9 or ahomologue thereof or a part of the gene; and redifferentiating thetransformed plant cell to obtain a plant, wherein the DS9 or thehomologue thereof is an ATP-dependent Zn-type metalloprotease, and thepolynucleotide decreases or increases production of the ATP-dependentZn-type metalloprotease in the plant cell, whereby cell death of a cellin the plant is promoted or suppressed.

[0012] A polynucleotide containing a gene encoding DS9 or a homologuethereof or a part thereof may be incorporated into a DNA in a nucleus ofa plant cell by a known gene recombinant technique. The term“polynucleotide” refers to a polymer of nucleotides, and is not limitedto a particular chain length.

[0013] In one embodiment of the present invention, the polynucleotidecontains the gene encoding the DS9 or the homologue thereof or the partof the gene in an antisense orientation, whereby cell death of a cell inthe plant is promoted.

[0014] A method for producing a plant which is conferred with resistanceto environmental stress of the present invention includes the steps of:transforming a plant cell with a polynucleotide containing a geneencoding DS9 or a homologue thereof or a part of the gene; andredifferentiating the transformed plant cell to obtain a plant, whereinthe DS9 or the homologue thereof is an ATP-dependent Zn-typemetalloprotease, and the polynucleotide decreases or increasesproduction of the ATP-dependent Zn-type metalloprotease in the plantcell.

[0015] In one emboidment of the present invention, the environmentalstress is pathogen infection.

[0016] In another embodiment of the present invention, thepolynucleotide contains the gene encoding the DS9 or the homologuethereof or the part of the gene in an antisense orientation.

[0017] In another embodiment of the present invention, the homologue hasa homology of about 70% or more with respect to an ATPase region of theDS9.

[0018] In a method for screening a selective inhibitor of a geneencoding DS9 or a homologue thereof of the present invention, the DS9 orthe homologue thereof is an ATP-dependent Zn-type metalloprotease,wherein the method includes the steps of: introducing a candidateinhibitor into an expression system having a gene encoding the DS9 orthe homologue thereof; and identifying whether or not production of theDS9 or the homologue thereof is selectively decreased in the expressionsystem.

[0019] Thus, the invention described herein makes possible theadvantages of (1) providing a method for promoting or suppressing celldeath by regulating an expression level of a cell death regulatory gene;and (2) providing a method for producing a plant which is conferred withresistance to environmental stress, e.g., pathogen infection, byregulating cell death; and (3) providing a method for screening aselective inhibitor of a cell death regulatory gene.

[0020] These and other advantages of the present invention will becomeapparent to those skilled in the art upon reading and understanding thefollowing detailed description with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a schematic diagram showing a structural comparisonbetween DS9 and other ATP-dependent Zn-type metalloproteases. The DS9includes a hydrophobic region on the N-terminus, an ATPase region in themiddle, and a Zn²⁺ binding motif on the C-terminus, as in the otherATP-dependent Zn-type metalloproteases. The hydrophobic region isconsidered to be a trans-membrane region.

[0022]FIG. 2 is a graph showing Mg²⁺-dependent ATPase activity ofGST-DS9 fusion protein.

[0023]FIG. 3 is an electrophoresis photograph showing the results ofnorthern analysis for NN tobacco infected with TMV. This analysisexhibits fluctuations in DS9 transcription after the temperature shiftfrom 30° C. to 20° C. As a control for fluctuations, wounded leaves wereused as a mock. mRNA was measured as a control for expression. As acontrol for showing degree of the HR, PR-1 gene was measured. PR-1 geneis specifically expressed during infection. Each number on the top ofthe figure denotes the time after the temperature shift from 30° C. to20° C.

[0024]FIG. 4 is an electrophoresis photograph showing the results ofwestern blotting analysis for NN tobacco infected with TMV. Thisanalysis exhibits fluctuations in the amount of DS9 protein after thetemperature shift from 30° C. to 20° C. As a control for fluctuations,wounded leaves were used as a mock. Anti-DS9 antibody was used afor theanalysis. Each number in the upper portion of the figure denotes thetime after the temperature shift from 30° C. to 20° C. Each number inthe lower portion of the figure denotes the amount of the protein ateach time, with the amount of the protein at the 0th time after the mockinfection being 100%.

[0025]FIG. 5 is an electrophoresis photograph showing the results ofnorthern analysis and western blotting analysis for NN tobacco infectedwith TMV after treatment with AMD and heat. These analyses exhibit DS9fluctuations in transcription level and protein amount.

[0026]FIGS. 6A to 6D are electron microscope photographs showingchloroplast localization of DS9.

[0027]FIG. 6A is an electron microscope photograph showing DS9 proteinlocalization in a mesophyll cell treated with an anti-DS9 antibody andanti-rabbit IgG conjugated with 10 nm-gold particles. A horizontal barin the figure represents 1 μm.

[0028]FIG. 6B is an electron microscope photograph showing FIG. 6A in ahigher magnification. The horizontal bar represents 0.1 μm.

[0029]FIG. 6C is an electron microscope photograph of a mesophyll celltreated with non-immunized serum (control) and anti-rabbit IgGconjugated with 10 nm-gold particles. The horizontal bar represents 0.1μm.

[0030]FIG. 6D is an electron microscope photograph showing DS9localization in a frozen section of a mesophyll cell treated with ananti-DS9 antibody and anti-rabbit IgG conjugated 10 nm-gold particles.The horizontal bar represents 0.1 μm.

[0031]FIG. 7 shows photographs of morphology representing necroticlesions in tobacco treated as follows: incubating NN and nn tobaccoinfected with TMV at 30° C. for 40 hours; treating the tobacco withmetalloprotein inhibitor (EDTA) and, chloroplast electron-transportchain inhibitor (DCMU); and further incubating the tobacco at 30° C. for24 hours.

[0032]FIG. 8 is an electrophoresis photograph showing the results ofnorthern analysis representing expression levels of PR-1 in tobaccotreated as follows: incubating NN and nn tobacco infected with TMV at30° C. for 40 hours; treating the tobacco with metalloprotein inhibitor(EDTA) and chloroplast electron-transport chain inhibitor (DCMU); andfurther incubating the tobacco at 30° C. for 24 hours.

[0033]FIG. 9 is a diagram showing the amount of salicylic acid intobacco treated as follows: incubating NN and nn tobacco infected withTMV at 30° C. for 40 hours; treating the tobacco with metalloproteininhibitor (EDTA) and chloroplast electron-transport chain inhibitor(DCMU); and further incubating the tobacco at 30° C. for 24 hours.

[0034]FIG. 10 is a graph showing the decrease in chloroplast functionwhen the HR is induced, measured using PSII activity as an index.

[0035]FIG. 11 is an electrophoresis photograph showing the results ofwestern blotting analysis for tobacco with a DS9 gene introduced theretoin a sense (S) or antisense (A) orientation. This analysis representsthe amount of DS9 protein in the transgenic tobacco. Anti-DS9 antibodywas used for the analysis. Wild-type tobacco was used as a controlregarding the amount of protein.

[0036]FIG. 12 is a graph showing diameters of necrotic lesions intobacco with a DS9 gene introduced thereto in a sense (S) or antisense(A) orientation. The necrotic lesions were obtained 5 days after TMVinoculation in the transgenic tobacco. Wild-type tobacco was used as acontrol.

[0037]FIG. 13 is a photograph of morphology representing necroticlesions in tobacco (A9 and S6) with a DS9 gene introduced thereto in asense (S) and antisense (A) orientation. The necrotic lesions wereobtained 5 days after TMV infection of the transgenic tobacco. Wild-typetobacco was used as a control.

[0038]FIG. 14 is a photograph of morphology of a transgenic tobacco (A9)with a DS9 gene introduced thereto in an antisense orientation. Thetobacco was observed 7 days after infection with Zhizoctonia solan. As acontrol, a transgenic tobacco with 35S-GUS introduced thereto was used.

[0039]FIG. 15 is a photograph showing morphology of transgenic tobacco(S4) with a DS9 sense gene introduced thereto. The tobacco was observedafter paraquat treatment. Wild-type tobacco (WT) was used as a control.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] DS9 is one of ATP-dependent Zn-type metalloprotease newlyisolated from a higher plant. The inventors have shown that DS9 andhomologues thereof are a cell death-regulating factor. This regulationis conducted, for example, by suppressing production of DS9 or ahomologue thereof under environmental stress, which results in inductionof cell death of a plant cell. The present invention is based on thisnovel finding.

[0041] The inventors isolated 6 clones which are expressed in a mannerspecific to the occurrence of the HR in tobacco. One of the isolatedclones was designated as a “DS9 gene”, and its entire base sequence wasdetermined.

[0042] Based on a deduced amino acid sequence encoded by the DS9 gene, ahomologous gene was searched. As a result, the DS9 amino acid sequencewas found to show 40%, 30%, 80%, and 42% homology with the respectiveamino acid sequences of the following ATP-dependent Zn-typemetalloproteases: FtsH from E. coli, Osdlp from yeast, ArFtsH fromArabidopsis, and Pftf from red pepper. In particular, a high homologywas found in a conserved region specific to ATPase. Table 1 shows thecomparison in the amino acid sequence among DS9 used in the presentinvention, FtsH from E. coli, Osdlp from yeast, ArFtsH from Arabidopsis,and Pftf from red pepper. Each amino acid is represented by the oneletter code. TABLE 1                       Walker motif A                       {overscore (|        |)} DS9  KNPDKYTALGAKIPKGCLLV GPPGTGKTLL ARAVAGEAGV PFFSCAASEF VELFVGVGAS 332 ArFtsH .......... .......... .......... .......... ....SRPQ.. .......... 341FtsH  RE.SRFQK.. G.....V.M. .......... .K.I....K. ...TISG.D. ..M.......231 Osd1p  .D.T..ES.. G.L...V..T .......... ...T...... D..FMSG...D.VY.....K 360 Pftf  .K.ERF..V. .R....V... .......... .K.I..........ISG... ..M....... 309                  Walker motif B                  {overscore (|          |)} DS9  RVRDLFEKAK SKAPCIVFIDEIDAVGRQRG AGMGGGNDER EQTINQLLTE MDGFSGNSGV 392 ArFtsH  .................... .......... .......... .......... .......... 401 FtsH ....M..Q.. KA....I... .......... ..L...H... ......M.V. ....E..E.I 291Osd1p  .I....AQ.R .R..A.I... .L..I.GKRN P---KDQAYA K..L....V. L....QT..I417 Pftf  ......K... EN......V. .......... T.I....... ...L..........E..T.I 369              SRH      {overscore(|                  |+E,OVS )} DS9  IVLAATNRPD VLDSALLRPG RFDRQVTVDRPDVAGRIKIL QVHSRGKALA KDVDFEKIAR 452 ArFtsH  .......... .................... ......V... .........G .....D.V.. 461 FtsH  ..I..........F...... ......V.GL ...R..EQ.. K..M.RVP.. P.I.AAI... 351 Osd1p .IIG...F.E A..K...... ...KV.N..L ...R..AD.. KH.MKKIT.. DN..PTI... 477Pftf  ..V.....A. I......... ......S..V ..IK..TE.. K..AGN.KFD S..SL.V..M429 DS9  RTPGFTGADL QNLMNEAAIL AARRELKEIS KNEISDALER IIAGPEKKNAVVSEEKKKLV 512 ArFtsH  .......... .......... ....DV.... .D.................. .......R.. 521 FtsH  G....S.... A..V....LF ...GNKRVV.MV.FEK.KDK .MM.A.RRSM .MT.AQ.EST 411 Osd1p  G...LS..E. A..V.Q..VY.CQKNAVSVD MSHFEW.KDK .LM.A.R.TM .LTDAARKAT 537 Pftf  .....S....A..L...... .G..GKTA.A SK..D.SID. .V..M.-GTV MTDGKS.S.V 488 Zn-bindingmotif    {overscore (     |)} DS9  AYHEAAHALV GALMPEYDPV PKISIIPRGQAGGLTFFAPS EERLESGLYS RSYLENQMAV 572 ArFtsH  .....G.... ..........A......... ......... ......... ......... 581 FtsH  .....G..II .R.V..H...H.VT.....R .L.V...L.E GDAISA---. .QK..S.IST 468 Osd1p  .F...G..IMAKYTNGAT.L YKAT.L...R .L.I..QL.E MDKVDI---T KRECQARLD. 594 Pftf .Y..VG..IC .TLT.GH... Q.VTL..... .K...W.I.A DDPTLI---. KQQ.FARIVG 545

[0043] As in the other ATP-dependent Zn-type metalloproteases, the DS9includes hydrophobic regions (in Table 1, represented by “Walker motifA” and “Walker motif B”), which are considered to penetrate a membrane,near the N-terminus, an ATPase region (represented by “SRH”) in themiddle portion, and a Zn²+binding motif near the C-terminus (FIG. 1).Furthermore, the DS9 recombinantly expressed in E. coli actuallyexhibited ATPase activity (FIG. 2). These results show that the DS9 usedin the present invention is an ATP-dependent Zn-type metalloprotease.

[0044] As a result of analysis on the function of the DS 9 gene when theHR was induced, it was shown that the DS9 gene was suppressed until theHR occurred, for both the transcription level and the amount of aprotein that is a translation product (FIGS. 3 and 4). In a leaf of NNtobacco infected with TMV, the transcription level and the amount of theprotein were decreased within one hour after the temperature shift from30° C. to 20° C. On the other hand, in a leaf subjected to mockinfection, the transcription level of the DS9 gene and the amount ofprotein were constant (see Examples 4 and 6). Furthermore, when the leafsubjected to mock infection were treated with actinomycin D (AMD) andheat shock (HS) which are known to induce the HR, the amount of the DS9protein was remarkably decreased (see Example 7).

[0045] Based on the above experimental results, the inventors confirmedthat the DS9 is a factor having a function of regulating cell death in aplant, and conducted various experiments for the purpose of developingthe method for using the DS9. As a result, the following was found.

[0046] 1) As a result of testing the effects of various proteaseinhibitors in inducing the formation of necrotic lesions, it was shownthat cell death was induced in TMV-infected tobacco only in the case ofusing a metalloprotease inhibitor.

[0047] 2) The DS9 was found to be localized in chloroplast.

[0048] 3) The HR was induced in cases of both the metalloproteaseinhibition and the decrease in a function of chloroplast, even when atemperature was not shifted in a manner known to be needed for the HR tooccur.

[0049] 4) The decrease in a function of the chloroplast, which wasobserved upon induction of the HR, was related to the decrease in theamount of the DS9 protein.

[0050] 5) Cell death was promoted in a plant in which the DS9 gene wasintroduced in an antisense orientation so as to decrease thetranscription level of the DS9 gene and the amount of the protein. Thisantisense plant acquired resistance to pathogen.

[0051] 6) Cell death was suppressed in a plant in which the DS9 gene wasintroduced in a sense orientation so as to increase the transcriptionlevel of the DS9 gene and the amount of protein. This sense plantacquired resistance to a superoxide-generating herbicide.

[0052] FtsH, a homologue of the DS9, is derived from a bacterium. It isinteresting that the DS9 was found in chloroplast, which is consideredto be derived from a bacterium, in terms of considering its origin. Itis reported that ArFtsH, another homologue of the DS9, is also localizedin chloroplast (Lindahl et al., The Journal of Biological Chemistry,Vol. 271, pp. 29329-29334 (1996)). Thus, a possibility is suggested thata homologue of the DS9 generally functions in chloroplast. Furthermore,it is assumed that the DS9 and a homologue thereof function in amitochondrion which is also considered to be derived from a bacterium.Actually, it is shown that a FtsH homologue is involved in decompositionof unfolded subunit 2 of cytochrome C oxidase in yeast mitochondria (T.Nakai et al., Mol. Cell. Biol., 15, 4441-4452 (1995)).

[0053] It is reported that in mammals, apoptosis is caused by thedecrease in a membrane electric potential due to the inhibition of anelectron-transport system in mitochondria. The decrease in the membraneelectric potential is inhibited by Bcl-2 or the like which is theproduct of a cell death regulatory gene(N. Zamzami et al., Exp. Med.,182, 367-377 (1995)). Furthermore, it is reported that cell death iscaused in the case where an electron-transport system does notsuccessfully function in mitochondria in mammals (Kripper et al., TheJournal of Biological Chemistry, 271, 21629, (1996) and Quillet-Mary etal., The Journal of Biological Chemistry, 272, 21388, (1997)). On theother hand, it is reported that a certain kind of protease works tomaintain homeostasis in chloroplast (Zatch Adam, Plant MolecularBiology, 32, 773-783, (1996)).

[0054] Considering the above, the mechanism of cell death which isregulated in the present invention can be explained as described below.It should be noted that the scope of the method of the present inventionis not limited or bound by the following mechanism.

[0055] The DS9 or a homologue thereof, which is a metalloprotease,decomposes unfolded proteins or abnormal proteins, thereby maintaininghomeostasis of chloroplast and mitochondria. When a plant is placedunder environmental stress, transcription of a gene encoding the DS9 ora homologue thereof in chloroplast and mitochondria is suppressed. As aresult, in some tissues, the protein level of a translation product (andthus activity level of the metalloprotease) decreased. In these tissues,cell death is induced as shown by the inventors. That is, when unfoldedproteins or abnormal proteins increase and accumulate in the chloroplastand mitochondria, they serve to decrease the function (i.e., ATP orNADPH production) of chloroplast and mitochondria. In chloroplast, wherephotosynthesis is conducted, light energy cannot be processedsuccessfully in chloroplast due to the decrease in this function. As aresult, active oxygen is generated in a plant cell. Such a collapse ofhomeostasis in a cell (i.e., collapse of an electron-transport systemand resultant accumulation of active oxygen) eventually leads to celldeath. It is considered that the DS9 or a homologue thereof decomposesthe above-mentioned unnecessary proteins to save cells from death andfunctions so as to maintain homeostasis.

[0056] Accordingly, in view of various factors as mentioned above, it isexpected that the similar mechanism of cell death may exist not only inplant cells, but in eucaryocytes in general.

[0057] Hereinafter, the present invention will be described in moredetail.

[0058] As described above, the inventors revealed a function of a celldeath regulatory gene which promotes or suppresses cell death dependingupon its expression level. The present invention is based on thisfinding.

[0059] According to the present invention, a method for promoting orsuppressing cell death by regulating an expression level of a cell deathregulatory gene is provided. Furthermore, according to the presentinvention, a method for producing a plant which is conferred withresistance to various environmental stress by regulating cell death isprovided.

[0060] The term “cell death regulatory gene” as used herein refers to agene which promotes or suppresses cell death depending upon itsexpression level. According to the present invention, a gene encodingthe DS9 and a homologue thereof and a part of the gene are contemplatedto be the cell death regulatory genes.

[0061] As used herein, the term “expression” of a gene refers totranscription of DNA into mRNA. The degree of transcription to mRNA isrepresented by an expression level. Thus, suppression of transcriptionrefers to the decrease in an expression level, and promotion oftranscription refers to the increase in an expression level.

[0062] The term “DS9” as used herein refers to an ATP-dependent Zn-typemetalloprotease having an amino acid sequence represented in SEQ.ID NO.1 of Sequence Listing. The term “ATP-dependent Zn-type metalloprotease”refers to a protease which requires ATP for its enzyme activity andcontains a divalent metal ion (typically, Zn²⁺) in an active center.This enzyme includes, in its amino acid sequence, one or morehydrophobic region near the N-terminus, a metal ion binding region(typically, a Zn²⁺ binding region) near the C-terminus, and an ATPaseregion in the middle portion. Typically, the enzyme includes twohydrophobic regions near the N-terminus and a Zn²⁺ binding region nearthe C-terminus.

[0063] The term “homologue of the DS9” as used herein refers to anATP-dependent metalloprotease similar to the DS9, which is a proteinhaving a homology of at least about 30%, preferably at least about 40%,with respect to the entire amino acid sequence of the DS9. Furthermore,the term “homologue of the DS9” refers to a protein having a homology ofat least about 60%, preferably at least about 70% with respect to theamino acid sequence in the ATPase region of the DS9. Examples of thehomologue include FtsH from E. coli, Osdlp from yeast, ArFtsH fromArabidopsis, and Pftf from red pepper.

[0064] As a method for isolating a naturally occurring gene encoding theDS9 and a homologue thereof, for example, a differential screeningeffective for cloning a gene which expresses a variable amount of mRNAcan be used. A method for producing a gene library for conductingdifferential screening, stringent conditions used for hybridization witha probe, and a method for cloning a gene are well-known to those skilledin the art. For example, see Maniatis et al., Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York (1989).

[0065] A novel gene encoding a homologue of the DS9 can also be used asa cell death regulatory gene. Such a novel gene can be obtained fromvarious gene libraries of organisms, using a gene encoding the DS9 or aknown homologue thereof, or a fragment of the gene as a probe. Forexample, a gene library of a plant, a gene library of E. coli, and agene library of yeast can be used. Stringent conditions for screening alibrary are appropriately selected by those skilled in the art.

[0066] It can be easily determined whether or not an amino acid sequenceencoded by the obtained gene corresponds to a homologue of the DS9. Suchdetermination can be carried out by aligning the amino acid sequenceencoded by the obtained gene with the amino acid sequence of the DS9 byusing commercially available computer analysis software (e.g., GeneWorks (IntelliGenetics, Inc.)), and investigating the homologytherebetween.

[0067] As a gene encoding the DS9 or a homologue thereof, or a part ofthe gene, an artificially synthesized gene or a part thereof, as well asa naturally occurring gene can be used. Hereinafter, the subject genewill be interchangeably referred to as a “DS9-related gene”.

[0068] The term “a part thereof” as used herein refers to a fragment ofthe gene used in the present invention having a length sufficient forreproducing the cell death regulatory function of the DS9 or a homologuethereof.

[0069] The term “a part thereof” in the case where a DS9-related gene isintroduced in an antisense orientation refers to a fragment havingcomplementation and length sufficient for an antisense RNA produced tocontain the said part to inhibit translation of mRNA which is a sensestrand in a plant. The fragment has a homology, as a sense strand, oftypically about 50%, preferably about 80%, more preferably about 90%,and most preferably about 95% or more in a nucleotide sequence levelwith a region complementary to an endogenous DS9-related gene present inthe plant. The length of the fragment is typically at least about 20nucleotides, preferably at least about 50 nucleotides, more preferablyat least about 100 nucleotides, and most preferably at least about 200nucleotides.

[0070] The term “a part thereof” in the case where a DS9-related gene isintroduced in a sense orientation refers to a fragment of the geneincluding a sequence encoding a region sufficiently extended, such thatan expression product, i.e., a part of the DS9 or a homologue thereofexhibits metalloprotease activity. This fragment preferably encodes ametal ion binding region and an ATPase region of a metalloprotease. Morepreferably, the fragment further encodes a signal region for localizingthe expression product in a cell organelle of interest.

[0071] A DS9-related gene or a part thereof can be introduced into aplant through an appropriate plant expression vector integrated in asense or antisense orientation depending upon the purpose of regulationof cell death. In the case where a DS9-related gene or a part thereof isintroduced in an antisense orientation, cell death is typicallypromoted. In the case where a DS9-related gene or a part thereof isintroduced in a sense orientation, cell death is typically suppressed.However, it is well-known in the art that co-suppression may occurdepending upon the expression level of an introduced gene. Theco-suppression is a phenomenon in which, as a result that mRNA of anintroduced gene is excessively produced, the expression level of theintroduced gene and a homologous, endogenous gene are both suppressed.When the co-suppression occurs, the expression level of a DS9-relatedgene may be suppressed, thereby promoting cell death.

[0072] As is appreciated by those skilled in the art, in the case wherethe co-suppression allows the effects of the present invention to beobtained, the above-mentioned “part thereof” introduced in a senseorientation may not be needed to encode a region sufficient forexhibiting metalloprotease activity. In this case, the definition of the“part thereof” with respect to the antisense orientation is applied.

[0073] The “plant” to which the method of the present invention isapplied includes both monocotyledon and dicotyledon. Examples of theparticularly preferable plants include tobacco, green pepper, eggplant,melon, tomato, sweet potato, cabbage, spring onion, broccoli, carrot,cucumber, citrus fruit, Chinese cabbage, lettuce, peach, rice, potato,barley, flour, and apple. Unless otherwise indicated, a plant as usedherein includes any one of a plant body, a plant organ, a plant tissue,a plant cell, and a seed. Examples of the plant organ include a root, aleaf, a pedicle, and a flower. Examples of the plant cell include callusand a suspension culture cell.

[0074] It may be preferable, but not required, that a cell deathregulatory gene which can be used in the method of the present inventionis derived from a plant of the same species as, or of species relatedto, that of a plant of interest (e.g., species classified into the samegenus or family).

[0075] The term “polynucleotide” as used herein has a DS9-infected geneor a part thereof, and any additional sequence required for achievingdesired transformation. The polynucleotide is typically in the form of aplant expression vector.

[0076] The “plant expression vector” as used herein refers to arecombinant construct of a nucleic acid sequence in which variousregulatory elements, such as a promoter for regulating the expressionlevel of a gene of interest, are linked to the gene or to each other insuch a manner as to be operable in a host plant cell. Preferably, theplant expression vector may include a plant promoter, a terminator, amarker gene such as a drug resistant gene, and an enhancer. Morepreferably, the plant expression vector may include an origin ofreplication. It is well-known to those skilled in the art that the typeof a plant expression vector and the preferable kind of a regulatoryelement may be varied depending upon a host cell.

[0077] According to the present invention, those skilled in the art canregulate the degree of cell death by appropriately selecting aregulatory element such as a promoter and an enhancer.

[0078] The plant expression vector used in the present invention mayfurther contain a T-DNA region. The T-DNA region allows a gene to beefficiently introduced into a plant genome, especially whenAgrobacterium is used to transform a plant.

[0079] The term “plant promoter” as used herein refers to a promotercapable of functioning in a plant cell. Examples of the plant promotersinclude, but are not limited to, promoters whose expressions are inducedby a certain kind of stress, for example, a promoter of a gene encodingan infection specific protein PR-1 of tobacco (hereinafter, referred toas “tobacco PR-1 promoter”) and promoters whose expressions are inducedby heat shock, or constitutive promoters such as a Cauliflower mosaicvirus (CaMV) 35S promoter and a promoter of nopaline synthase (Pnos).

[0080] The term “terminator” as used herein refers to a sequencepositioned downstream of a region of a gene encoding a protein, which isinvolved in the termination of transcription of mRNA, and the additionof a poly A sequence. The terminator is known to contribute to thestability of mRNA, thereby affecting the expression level of a gene.Examples of the terminator include, but not limited to, a CaMV 35Sterminator, a terminator of a nopaline synthase gene (Tnos), and aterminator of a tobacco PR-1 gene.

[0081] The “drug resistance gene” is desirable to facilitate theselection of the transgenic plant. As the drug resistance gene, aneomycin phosphotransferase II (NPTII) gene for conferring kanamycinresistance, a hygromycine phosphotransferase gene for conferringhygromycine resistance, and the like are preferably used.

[0082] Examples of promoters for expressing the drug resistance geneinclude, but are not limited to, the above-mentioned plant promoterssuch as a tobacco PR-1 promoter, a CaMV 35S promotor, and a nopalinesynthase promoter.

[0083] An enhancer may be used to enhance expression of a gene ofinterest. As the enhancer, an enhancer region containing a sequenceupstream of the above-mentioned CaMV 35S promoter is preferable. Aplurality of enhancers may be used per one gene of interest.

[0084] A vector used in the present invention for constructing a plantexpression vector may preferably be a pBI-type vector, a pUC-typevector, or a pTRA-type vector.

[0085] The pBI-type and pTRA-type vectors may introduce a gene ofinterest, using Agrobacterium, into a plant. A pBI-type binary vector oran intermediate vector may be preferably used. Examples of the pBI-typevector include pBI121, pBI101, pBI101.2 and pBI101.3. These vectorscontain a gene from a T-DNA region, which can be introduced into a plantvia Agrobacterium mediated transformation. These vectors also contain aNPTII gene which confers kanamycin resistance to a plant. The NPTII geneis expressed under the control of a plant promoter to serve as a markergene.

[0086] Use of the pUC-type vector may allow a gene to be directlyintroduced into a plant. Examples of the pUC-type vector include pUC18,pUC19 and pUC9.

[0087] The plant expression vector of the present invention can beproduced by using a gene recombinant technique well-known to thoseskilled in the art. Preferably, a DS9-related gene or a part thereof isincorporated downstream of a promoter of the vector in a sense orantisense orientation.

[0088] A plant expression vector is introduced into a plant cell byusing methods well-known to those skilled in the art, for example, amethod of infecting a plant cell with Agrobacterium or a method ofdirectly introducing a plant expression vector into a plant cell. As amethod for introducing a plant expression vector into a plant cell viaAgrobacterium, for example, the method of Nagel et al. (Micribiol.Lett., 67, 325 (1990)) can be used. According to this method, forexample, first, Agrobacterium is transformed with a plant expressionvector by, for example, electroporation, and then, the transformedAgrobacterium is introduced into a plant cell in accordance with amethod described in Plant Molecular Biology Manual (S. B. Gelvin et al.,Academic Press Publishers). Examples of the method for introducing aplant expression vector directly into a plant cell include anelectroporation method, a gene gun method, a calcium phosphate method,and a polyethylene glycol (PEG) method. These methods are well-known inthe art, and a method suitable for a particular plant to be transformedcan be suitably selected by those skilled in the art.

[0089] A cell transformed by introducing a plant expression vector maybe selected based on its drug resistance such as kanamycin resistance.Thereafter, the transformed cell can be regenerated as a plant tissue, aplant organ, and/or a plant body by using a conventional method.Furthermore, seeds can be obtained from the regenerated plant body.Accordingly, a plant having a cell death regulatory gene in its cellscan be obtained.

[0090] In the plant thus obtained, a DS9-related gene or a part thereofis expressed, whereby cell death of a cell in the plant can be promotedor suppressed.

[0091] In a plant in which production of the DS9 or a homologue thereof(i.e., production of a metalloprotease) is decreased, cell death isgenerally promoted. In a plant in which the tendency of causing celldeath is appropriately enhanced, resistance to a certain kind ofenvironmental stress, in particular, resistance to a pathogen due to theformation of necrotic lesions, can be exhibited. A primary mechanism forthis resistance may be explained as follows: cell death is locallypromoted, so as to enclose a pathogen growing in an infected region,whereby an infected site can be prevented from spreading. In a naturalplant, the mRNA and protein of the DS9 or homologue thereof may bedecreased in cells at a site subjected to pathogen infection, so thatonly the site causes cell death. In a transgenic plant produced so thatproduction of the DS9 or a homologue thereof is decreased, a cellinfected with a pathogen is more likely to die. Consequently, the celldeath of an infected cell is promoted (i.e., the transgenic plantbecomes resistance to the pathogen).

[0092] In a plant in which production of the DS9 or a homologue thereofis increased, cell death is generally suppressed. In a plant in whichthe tendency of causing cell death is appropriately reduced, resistanceto various environmental stresses can also be exhibited. When a plant isexposed to environmental stress, an endogenous DS9-related gene can beinactivated. As a result, it becomes difficult to maintain homeostasisof an organelle, and harmful active oxygen can be generated in a plantcell. According to the method of the present invention, production ofthe DS9 or a homologue thereof can be increased. This leads to anincrease in the activity of an metalloprotease, whereby a function ofmaintaining homeostasis of an organelle (e.g., mitochondria andchloroplast) is improved. Consequently, generation of active oxygen issuppressed to a low level, and cell death is suppressed.

[0093] The term “environmental stress” as used herein refers to anystress which may be given to plants in the natural environment toprevent growth thereof. Examples of the environmental stress includepathogen infection, strong light, low temperature, freezing, drying,high temperature, high salt concentration, UV irradiation, ozone, and aherbicide.

[0094] The term “conferred resistance” to environmental stress refers toproviding a new type of resistance to plants or enhancing the existingresistance of plants.

[0095] The term “pathogen infection” refers to infection with apathogenic factor of a plant, including infection with virus, viroid,filamentous fungi, and bacteria.

[0096] The presence of resistance to environmental stress can beconfirmed by evaluating the difference which can be observed between atransgenic plant and a control plant when the both plants are put undera certain environmental stress.

[0097] For example, disease resistance of a transgenic plant withrespect to pathogen infection can be evaluated as the difference in amorphology change between a transgenic plant and a control plant whenplants are infected with a pathogen (e.g., virus such as TMV, andfilamentous fungi such as Rhizoctonia solani). For example, in the casewhere the degree of necrotic lesions observed in the transgenic plantafter pathogen infection is significantly suppressed, compared with thecontrol plant, it is understood that the transgenic plant is conferredwith the resistance.

[0098] A transgenic plant which is conferred with resistance to pathogeninfection in accordance with the present invention includes plants whichare resistance at least one of TMV and Rhizoctonia solani.

[0099] Herbicide resistance of a transgenic plant can be evaluated asresistance to a known herbicide such as a superoxide-generatingherbicide (e.g., 1,1-dimethyl-4,4-bipyridinium dichloride; sold under atradename “paraquat”). For example, in the case where the degree ofdecomposition of chlorophyll a and chlorophyll b is significantlysuppressed in a transgenic plant after herbicide treatment, comparedwith a control plant, it is understood that the transgenic plant isconferred with the resistance.

[0100] As described above, it is suggested that regulation of cell deathby an ATP-dependent Zn-type metalloprotease, such as DS9, is deeplyinvolved in biofunction not only in the plants but also in theeucaryotes in general. Thus, a selective inhibitor of a DS9-related genemay be important as means for selectively suppressing biofunction. Forexample, such an inhibitor can be utilized as a candidate for anagrochemical or a pharmaceutical.

[0101] The screening of a selective inhibitor of a DS9-related gene isconducted by introducing a candidate inhibitor into a plant cellcontaining a DS9-related gene, and identifying whether or not productionof the DS9 or a homologue thereof is selectively decreased in the plantcell. The conditions for performing this screening method can beappropriately selected by those skilled in the art. The screening isperformed, for example, by inoculating tobacco having no N gene withTMV, followed by treatment with a candidate inhibitor, and investigatingwhether or not necrotic lesions are formed in the TMV-infected portion.In the case where necrotic lesions are formed only in the TMV-infectedportion of the treated leaf, and no significant change in morphology isrecognized in the treated leaf which is not infected with TMV, thecandidate inhibitor is determined to be selective for a DS9-relatedgene.

[0102] For example, a plant cell used for the screening can exist in theform of any type of a plant. Preferably, the plant cell is used in vitroas a free plant cell. The plant cell preferably has a DS9-related geneas an endogenous gene. A candidate inhibitor includes, but is notlimited to, proteins, nucleic acids, saccharides, and lipids.Appropriate means for delivering the candidate inhibitor to a plant cellcan be selected by those skilled in the art depending upon the kind ofan inhibitor.

[0103] Whether or not production of the DS9 or a homologue thereof isdecreased in a plant cell treated with a candidate inhibitor can beappropriately identified by the method well-known to those skilled inthe art. For example, it can be easily determined by western blottinganalysis by comparing the protein amounts of the DS9 or a homologuethereof between the plant cell treated with a candidate inhibitor andthe untreated plant cell. In the case where production of the DS9 or ahomologue thereof is significantly decreased in the treated plant cell,it is understood that the candidate inhibitor used is an inhibitor of aDS9-related gene.

[0104] Hereinafter, the present invention will be further described indetail by way of the illustrative examples. The restriction enzymes,plasmids and the like used in the following examples are available fromcommercial sources.

EXAMPLE 1 Isolation of DS9 Gene

[0105] N. tabacum cv. Samsun NN and Samsun nn were grown in atemperature-controlled greenhouse at 25° C. under 16 hr of light, at anintensity of 120 μE/m²/s. OM strain of TMV (Gene bank of NationalInstitute of Agrobiological Resources, Ministry of Agriculture, Forestryand Fisheries, Japan) was used in this example. For the temperatureshift assay, a tobacco leaf was inoculated with TMV by gentle rubbing ofthe upper epidermis of a leaf with a suspension in 10 mM phosphatebuffer (pH 7.0) containing the virus at an adequate concentration andwet Carborundum (#600, Kishida Chemicals., Osaka, Japan), and incubatedfor 30 min at room temperature to allow the virus to invade. After theinfected leaf was washed with water to remove Carborundum, the leaf wasput into a transparent plastic container which was transferred to anincubator held at 30° C. under 24 hr of light, at an intensity of 60μE/m²/s. After incubating for 48 hr, the container was transfered to anincubator held at 20° C., under 24 hr of light at an intensity of 60μE/m²/s. During the incubation, both at 20° C. and 30° C., water wassupplied to a petiole of the leaf by covering with wet tissue paper.

[0106] Differential screening was performed as described by Seo et al.(Science, vol. 270, 1988, (1995)). Briefly, tobacco leaves were infectedwith TMV (10 μg/ml), incubated at 30° C. and then the tempereture wasshifted to 20° C. Poly(A)+RNA was isolated from the leaves harvested 3hr or 52 hr after the temperature shift. Two radioactively labelledsingle-stranded cDNA probes, i.e., a plus probe (prepared from thepoly(A)+RNA of 3 hr after the temperature shift) and a minus probe(prepared from the poly(A)+RNA of 52 hr after the temperature shift),were synthesized. These two probes were used to perform differentialscreening for a cDNA library. The library was prepared by a conventionalmethod from the poly(A)+RNA that were used for synthesis of the plusprobe. As a result, 6 clones were found to hybridize the plus probeonly. One cDNA was designated as DS9. This cDNA was excised with R408helper phage and recirclularized to subclone into a pBluescript SK⁻phagemid vector according to the manufacturer's instructions(Stratagene).

[0107] Both strands of DS9 were sequenced by the dideoxychain-termination method using a model 373A DNA sequencer (AppliedBiosystems). Nucleic acid and amino acid sequences were analyzed withthe GENE WORKS (IntelliGenetics) software system.

[0108] Amino acid sequences encoded by DS9 cDNA had 40%, 30%, 80% and42% homologies with the respective amino acid sequences of FtsH fromE.coli, Osdlp from yeast, ArFtsH from Arabidopsis thaliana and Pftf fromred pepper, respectively. Particularly high homologies were found in theATPase conserved regions. As with the other ATP dependent Zn-typemetalloproteases, DS9 had N-terminal hydrophobic regions considered tospan a membrane, a central ATPase region, and a C-terminal Zn²⁺-bindingmotif (FIG. 1).

EXAMPLE 2 Expression of Recombinant GST-DS9 Protein in E. coli

[0109] To generate GST-DS9 fusion gene, the DS9 coding region (fromposition 411 to 2240 in SEQ ID NO: 1) was amplified by PCR usingprimers: 5′-ACGTGGATCCTTGAATGCTGTGAAAAAGGGTA-3′ and5′-ACGTGAATTCTTATGCCTATTTCTCTTGCATC-3′. A BamHI-EcoRI fragment wassubcloned into the BamHI and EcoRI sites of pGEX-2T (Pharmacia; Smithand Johnson, Gene, 67, 31 (1988)) so that the DS9 is fused to the Cterminus of the GST protein. The resulting construct was sequencedaround the linkage site between GST and DS9 regions to confirm that therespective coding regions was linked in-frame. Because almost all of theGST-DS9 fusion protein was produced as an insoluble protein in E. coli,the protein was purified from the insoluble fraction by the followingprocedures.

[0110] The pGEX-DS9 was expressed in E. coli strain JM109 (Stratagene)by incubating with 0.4 mM IPTG for 12 hr. Cells were pelleted, washedand suspended in buffer A (20 mM Tris-HCl, pH 8.0, 30 mM NaCl, 10 mMEDTA, 2 mM phenylmethanesulfonyl fluoride (PMSF)). After addition of{fraction (1/10)} volume of lysozyme (20 mg/ml in buffer A), thesuspension was incubated for 1 hr on ice and cells were disrupted bysonication. The insoluble fraction was collected by centrifugation at6,000× g for 10 min at 4° C., washed three times with buffer B (20 mMTris-HCl, pH 7.5, 30 mM NaCl). The insoluble fraction was then collectedby centrifugation at 8,000× g for 10 min at 4° C., and resuspended in 10mM EDTA (pH 8.0).

[0111] After addition of 8M guanidine-HCl (pH 8.3) to a finalconcentration of 6.22M, the lysate was subjected to centrifugation at12,000× g for 30 min at 4° C. The supernatant was dialyzed first againstbuffer C (2M guanidine-HCl, 0.2 mM EDTA, pH 8.0, 5 mM β-mercaptoethanol)for 2 hr at 4° C., then against buffer D (20 mM Tris-HCl, pH 7.5, 100 mMNaCl, 0.5 mM EDTA, 5 mM β-mercaptoethanol) for more than 4 hr at 4° C.After collection by centrifugation at 100,000 X g for 30 min at 4° C.,the supernatant was subjected to affinity purification withglutathione-agarose beads according to the method of Smith and Johnsonet. al. (supra). Protein concentrations were determined by a Coomassiedye-based protein assay kit (Bio-Rad).

EXAMPLE 3 ATPase Activity Assay

[0112] In order to investigate whether DS9 has ATPase activity, theATPase activity was measured at 37° C. as described by Armon et. al.,(The Journal of Biological Chemistry, 265, 20723 (1990)). To assay theATP hydrolysis, the reaction mixture (25 μl) contained followingcomponents: 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 2 mM dithiothreitol(DTT), 1 mM ATP, 1 mM [α-32P]ATP (about 148 TBq/mmol; ICN BiomedicalsInc.), and 6 μg/ml GST-DS9 protein. Radioactivity was determined by aconventional method according to Cerenkiv radiation.

[0113] The results are shown in FIG. 2. It was thus shown that therecombinant protein expressed in E. coli had ATPase activity.

EXAMPLE 4 Fluctuation in the DS9 Transcriptional Level after theTemperature Shift

[0114] Northern analysis was performed to study fluctuation in the DS9transcription level after the temperature shift in TMV-infected NNtobacco.

[0115] A leaf of tobacco (N. tabacum cv. Samsun NN) was infected withTMV as described in Example 1 and incubated at 30° C. for 40 hr prior tothe temperature shift to 20° C. A leaf that had not been infected withTMV but had been wounded was used as a control (mock).

[0116] The leaves were collected at 0, 1, 3, 4, 8 and 24 hr after thetemperature shift to 20° C., and respective total RNAs were preparedaccording to the method of Seo et al. (supra).

[0117] For the DS9 cDNA, the partial DNA fragment corresponding to the3′ noncoding region of the cDNA was used as a probe.

[0118] As control probes, cDNAs encoding acidic PR-1 protein and basicPR-1 protein, which are expressed specifically upon infection were used.A cDNA probe encoding an acidic PR-1 protein was synthesized by PCR asdescribed by Matsuoka et al. (Plant Physiology, 85, 942 (1987)) usingprimer A: 5′-TACTAATTGAAACGACCTACGTCC-3′; primer B:5′-ATAATAATATCTGATCATACATCAAGC-3′ according to the conventional method.A cDNA encoding a basic PR-1 protein (Eyal et al., Plant MolecularBiology 19, 589 (1992)) was synthesized by PCR using synthetic primers(primer A: 5′-ATCCCTTTGATTCCAAGGTTGG-3′; primer B:5′-CAAAACACATACATATACACACCTCC-3′), which were designed from the reportedsequence data, according to the conventional method.

[0119] Northern hybridization was performed as described in Seo et al.(supra). Each blot was exposed to XAR film (Kodak) at −80° C. for 48 hrusing Intensyfying Screen (Kodak). Relative intensity of the DS9transcript was determined with the NIH Image 1.61 (National Institute ofHealth) program.

[0120] The results are shown in FIG. 3. It was found that transcriptionof the DS9 gene was suppressed until the HR occurred. The expressionlevel of the DS9 mRNA in a TMV infected leaf decreased within 1 hr afterthe temperature shift from 30° C. to 20° C. No expression of the DS9mRNA was detected 8 hr after the temperature shift. On the other hand,transcription of the DS9 gene in a mock infected leaf was consistent andwas not suppressed.

Example 5 Production of an anti-DS9 Protein Antibody

[0121] For antibody production, two rabbits were subjected tointraperitoneal injection of the recombinant GST-DS9 protein obtained inExample 2 in an amount of 400 μg/rabbit. Three additional injections ofeach 100 μg/rabbit, were given at 7-day intervals. The antisera wereobtained 2 weeks after the last injection and the immunoglobulinfractions were purified by chromatography on a Protein A-sepharosecolumn (Pharmacia). The fractions containing anti-DS9 antibody weredialyzed against PBS (20 mM KH₂PO₄, 140 mM NaCl, pH 7.4). The antibodywas tested for cross reactivity with DS9 protein on immunoblotscontaining fractionated total protein of E. coli.

EXAMPLE 6 Fluctuations of a DS9 Protein Amount after the TemperatureShift

[0122] Western blotting analysis was performed to investigate thefluctuations of a DS9 protein amount after the temperature shift inTMV-infected NN tobacco.

[0123] A leaf of tobacco (N. tabacum cv. Samsun NN) was infected withTMV as described in Example 1 and incubated at 30° C. for 40 hr prior tothe temperature shift to 20° C. A leaf that had not been infected withTMV but had been wounded was used as a control (mock).

[0124] The leaves were collected at 0, 3, 4, 6 hr (at 0 and 6 hr for themocks) after the temperature shift to 20° C. A protein sample obtainedfrom the collected leaves by the conventional method was separated by 8%SDS-polyacrylamide gel and transferred to an Immobilon membrane(Millipore) in a solution containing 25 mM Tris, 192 mM glycine and 20%methanol. After blocking with 2% BSA in TBST (20 mM Tris-HCl, pH 7.5,150 mM NaCl), the membranes were incubated for 1 hr with the anti-DS9antibody which was prepared in Example 5 (dilution 1:3000). The membranewas extensively washed in TBS containing 0.05% Tween 20, and incubatedfor 30 min with goat anti-rabbit IgG conjugated with alkalinephosphatase(Organon Teknika Corp., Durham) (dilution 1:2000). Thereaction was visualized by hydrolysis of a substratetetrazolium-5-bromo-4-chloro-3-indolyl phosphate. Relative intensity ofthe DS9 protein was determined using NIH Image 1.61 (National Instituteof Health) program.

[0125] The results are shown in FIG. 4. At 4 and 6 hr after thetemperature shift to 20° C., the amount of each DS9 protein in the TMVinfected leaves decreased to 78% and 62%, respectively. The DS9 proteinmass was shown to decrease specifically during the process leading tothe occurence of the HR.

EXAMPLE 7 Fluctuation in the DS9 Transcription Level and the amount ofProtein in the TMV-infected NN Tobacco After Treatment with AMD and Heat

[0126] Prior to the treatments, tobacco leaves were detached, inoculatedwith TMV (10 μg/ml in a phosphate buffer (pH 7.0)) or mock inoculated(buffer alone), and incubated at 30° C. for 40 hr. For reagenttreatment, after the incubation, petioles of the leaves were put intovials containing 0.5 ml of an inhibitor solutions containing AMD (SigmaChemical Co., St. Louis. Mo., USA) as a 10% (v/v) methanol aqueoussolution. The solution was absorbed through the petioles, and sterilewater was added to the vials within 1 hr. For heat treatment, aTMV-infected NN tobacco was subjected to heat shock by treating at 50°C. for 2 minutes. Each of the treated leaves was then further incubatedat 30° C. Northern analysis and Western blot analysis were performed asdescribed in Examples 4 and 6. In addition, those treated with waterwere used as controls.

[0127] The results are shown in FIG. 5. The AMD and heat treatmentswhich are known to induce the HR, were shown to decrease significantlythe DS9 transcription level and the DS9 protein amount in TMV-infectedNN tobacco.

EXAMPLE 8 Effects of Various Protease Inhibitors on Induction ofNecrotic Lesion Formation

[0128] The following reagents were used as inhibitors: APMSF, Aprotininand 3,4-D as serine protease inhibitors, E-64 as a cysteine proteaseinhibitor, EDTA as a metalloprotease inhibitor, and leupeptin as aserine/cysteine protease inhibitor. Each inhibitor was prepared asdescribed below.

[0129] For EDTA (Wako Pure Chemicals. Ind. (Osaka, Japan), an aqueoussolution adjusted to pH 8.0 by NaOH was used.

[0130] For E-64 (Boehringer Mannheim, Germany), a 50 mM solution inmethanol/H₂O (1:1, v/v) was used as a stock solution and subsequentlydiluted in water to the concentrations indicated below.

[0131] For 3,4-D (Boehringer Mannheim, Germany), a 50 mM solution indimethyl sulfoxide was used as a stock solution and subsequently dilutedin water to the concentrations indicated below.

[0132] Leupeptin, Aprotinin, and APMSP were each used as an aqueoussolution.

[0133] In this example, EDTA was used at concentrations of 1 mM, 10 mMand 50 mM, and other inhibitors were used at concentrations of 0.01 mM,0.1 mM and 1 mM.

[0134] Prior to reagent treatment, tobacco leaves were detached,inoculated with TMV (8 μg/ml in a phosphate buffer (pH 7.0)) or mock(buffer alone), and incubated at 30° C. for 40 hr. After the incubation,petioles of the leaves were put into vials containing 0.5 ml ofinhibitor solutions. The solution was absorbed through the petioles andsterile water was added to the vials within 1 hr. The treated leaveswere then further incubated at 30° C. The results are shown in Table 2.TABLE 2 Effects of various protease inhibitors in induction of necroticlesion formation^(a) Tobacco cv. Inhibitor Type of inhibitor NN nn APMSFSerine protease − − Aprotinin Serine protease − − 3,4-D Serine protease− − E-64 Cysteine protease − − EDTA metalloprotease + + leupeptinSerine/cysteine protease − −

[0135] A necrotic lesion was induced in NN tobacco when treated withEDTA. A similar necrotic lesion was also observed in nn tobaccocontaining no N gene. A necrotic lesion was not observed when the othervarious protease inhibitors were used. These results show thatinhibition of the metalloprotease activity, including the activity ofthe DS9 protein, is sufficient to induce cell death in a plant.

EXAMPLE 9 Localization of DS9 within a Cell

[0136] (9-1) Immunoelectron Microscopy

[0137] Immunoelectron microscopy was performed basically as described inSuzuki and Kataoka (Journal of Histochemistry, 40, 379 (1992)) andTomoyasu et al. (Journal of Bacteriology, 175, 1352 (1993)), except thata leaf was cut into pieces of 1×1 mm and fixed with 0.1% glutaraldehydeand 4% paraformaldehyde in sodium cacodylate (pH 7.4) under vacuumconditions. Then, the leaf tissue was embedded in LR White resin (TheLondon Resin Co., London) and was cut at −20° C. with an ultramicrotome.A section was incubated with anti-DS9 antibody (dilution 1:250) and thenreacted with goat anti-rabbit IgG conjugated with 10 nm-gold(dilution1:100; Biocell Research Laboratories, Cardiff) for 30 min at 37° C.After the immunolabeling, a section was stained with uranyl acetate.

[0138] As the cytochemical control, specimen was incubated withnon-immunnized rabbit IgG.

[0139] For immunoelectron microscopy using ultra-thin frozen sections,some pieces of fixed tissues were infused using the method described inTokuyasu (Histochemical Journal, 21, 163, 1989) with a mixture of 20%polyvinylpyrrolidone (MW 10,000: Sigma) and 1.6M sucrose, frozen inliquid propane, and then cryo-sectioned. The section was immunogoldlabelled by the same procedure as for the above described sampleembedded with LR White, and adsorption-stained with polyvinyl alcohol(MW 10,000; Sigma). Samples were observed by a transmission electronmicroscope (H-7100, Hitachi, Japan).

[0140] 9-2 Imaging with Gold Particles

[0141] A negative of an electron micrograph was digitized by a flat bedscanner (GT-9000, Epson; 1800 dpi) and stored by TIFF format. Thedigitized images were normalized to enhance the contrast according tothe method of Fukui (Theoretical and Applied Genetics, 72, 27 (1986)) byAdobe Photoshop ver. 3.0 (Adobe Systems, Incorp.). The boundaries ofnuclei, chloroplasts, mitochondria and microsomes were traced for eachof the electron micrographs, and the areas of the organelles weredigitally measured. The number of gold particles were visually countedfor each of the organelles.

[0142] These results indicated that the DS9 is localized in chloroplasts(see FIG. 6).

EXAMPLES 10 Induction of HR by EDTA and DCMU Treatments

[0143] To perform an inhibitor treatment with NN tobacco and nn tobacco,EDTA, a metalloprotease inhibitor, and DCMU(3-(3,4-dichlorophenyl)-1,1-dimethylurea) (Sigma Chemical Co., St Louis,MO, USA), an inhibitor of electron transport in a photochemical systemII (PSII), were used as described in Example 8. An EDTA solution wasprepared as described in Example 8. The DCMU was dissolved in methanol.The concentration of the inhibitors were 5 mM and 100 mM, respectively.

[0144] After the treatment, leavs were further incubated at 30° C., thenexamined regarding the followig: formation of a necrotic lesion;expression of PR-1; presence of a marker gene for the HR protein; andaccumulation of salicylic acid. For expression of the PR gene, Northernanalysis was performed as described in Example 4. Salicylic acid wasquantitated as follows.

[0145] Free salicylic acid was extracted and quantitated as described byMalamy et al. (The Plant Cell, 4, 359 (1992)). HPLC analysis wasperformed on a pBondasphere, 5-μm C-18 (3.9 mm×15 cm) column maintainedat 40° C. Isocratic separation was conducted with 23% (v/v) methanol in20 mM sodium acetate (pH 5.0). Fluorescence detection was performed atusing a Model RF-550A (Shimazu, Japan) at 1 m/min. All data werecorrected for losses. The results are shown in FIGS. 7, 8 and 9. In bothNN tobacco and nn tobacco, both EDTA and DCMU induced the HR. It wasshown that the HR is induced by inhibition of the metalloprotease ordecrease in the chloroplast function.

EXAMPLE 11 Function of a Chloroplast during HR Induction

[0146] Kinetics of chlorophyll fluorescence induction were measured withpulse amplitude modulation fluorimeter (PAM-2000, Heinz Walz, Germany).TMV infection was performed as described in Example 1. A leaf sample washeld so that its surface has an angle of 60° with respect to thedirection to the light source, and half of the leaf was exposed to anactinic light (600 μE/m²/s). The kinetics of fluorescence induction wasrecorded on a portable computer installed with Data Acquisition Software(DA-2000, Heinz Walz). The results were shown in FIG. 10.

[0147] The PSII activity in a leaf of NN tobacco inoculated with TMV didnot decrease during incubation at 30° C. However, after 4 hr from thetemperature shift to 20° C., the PSII activity started decreasing. Thistime point is consistent with the time that the DS9 protein mass startsto decrease. The results show that decrease in the chloroplast functioncorrelates with decrease in the DS9 protein amount.

EXAMPLE 12 Generation of Transgenic Plants

[0148] The DS9 coding region (positions 21 to 2240 of SEQ NO: 1) wasamplified by PCR using primers: primer A, 5′-ACTATGGCCAATTCTCTCTC-3′ andprimer B, 5′-TTATGCCTATTTCTCTTGCATC-3′.

[0149] For a sense construct, the BamHI and SacI sites were linked atthe 5′ ends of primers A and B, respectively. For an anti-senseconstruct, the SacI and BamHI sites were linked at the 5′ ends ofprimers A and B, respectively.

[0150] The resultant PCR products were verified by DNA sequencing. Theproducts were digested with BamHI and SacI and then ligated, in thesense and antisense orientation relative to the CaMV 35S promoter, to abinary vector, pBI121 (Clontech), which had previously been digestedwith BamHI and SacI. The sense and antisense DS9 expression constructswere introduced into Agrobacterium tumefaciens LBA4404 (Ooms et al.,Gene, 14, 33 (1981)) by electroporation (Wen-Jun and Forde, Nucleic AcidResearch, 17, 8385 (1989)). Transformation of Samsun NN tobacco wasperformed by the leaf-disc cocultivation method (Horsch et al., Science,227, 1229 (1985)). Leaf discs were immersed in a bacterial solution,placed in an incubation medium (basal Murashige-Skoog (MS) medium with3% sucrose and B5 vitamins) containing naphthaleneacetic acid (100 μg/L)and benzyl amino purine (1 mg/L) for 2 days at 25° C. under continuousillumination of white fluorescence lamp, at an intensity of 120pE/m²/s.Laef discs were then transferred to the foregoing incubation mediumwhich further contain 500 μg/ml carbenicillin. After 2 days, leaf discswere transferred to a selection medium (incubation medium containing 500pg/ml carbenicillin and 100 μg/ml kanamycin).

[0151] For generation of a transformant which expresses a sense DS9gene, a plate containing leaf discs was incubated at 25° C. under 16 hrof light at an intensity of 120 μE/m²/s. For generation of atransformant which expresses an antisense DS9 gene, incubation wascarried our at 25° C. under 24 hr of light at an intensity of 10μE/m²/s. Shoots formed in kanamycin-containing medium were transferredto a hormones-less selection medium. After rooting, plantlets weretransferred to a pot containing soil.

EXAMPLE 13 Analysis of Cell Death Regulation in Transgenic Plant

[0152] (TMV Infection)

[0153] Strains of self-pollinated, second generation were obtained fromthe transgenic tobacco obtained in Example 12, in which the DS9 cDNA wasexpressed in sense or in antisense. With these strains, the proteinamount was analysed by performing Western blot analysis as described inExample 6. (sense plants; S1, S4, S5, S6, S9; antisense plant; A9, A12)(FIG. 11). In addition, each strain was infected with TMV as describedin Example 1, and the size of the lesion caused by the infection wasobserved (FIGS. 12 and 13). In all experiments, wild type tobacco wasused as a control.

[0154] In a sense strain (S6) which contains a two-fold amount of theDS9 protein compared to a wild type tobacco, the size of the necroticlesion reached two-fold the size of that in the wild type. On the otherhand, in an antisense strain (A9) which contains about half amount ofthe DS9 protein compared to a wild type tobacco, the size of necroticlesion was about a half compared to the wild type. A small necroticlesion indicates that cell death occurs immediately (i.e. cell death ispromoted), thereby spreading of infected plaques is prevented.

[0155] These results indicate that cell death is promoted in a cellwhich contains a smaller amount of the DS9 protein, and suppressed in acell which contains a larger amount of the DS9 protein. Furthermore, itis apparent that a transgenic tobacco which expresses a DS9 cDNA in anantisense orientation exhibited increased resistance against TMVinfection.

[0156] (Infection with Zhizoctonia Solan)

[0157] A seed of a self-pollinazed, second generation strain of A9 (atransgenic tobacco obtained in Example 12, which expresses a DS9 cDNA inan antisense orientation) and, as a control, a seed of a tobacco inwhich a 35S-GUS construct was introduced were obtained. These seeds wereeach plated in a 9 cm-diameter petri dish containing MS agar medium with50 μg/ml kanamycin (see Murashige, T. and Skoog, F. , “A reversed mediumfor rapid growth and bioassay with tobacco tissue cultures”, PhysiologiaPlantarum, vol. 15, pp. 473-497(1962)).

[0158] Zhizotonia solan was pre-cultured in PDA medium (39 μg/l Bacto(Trademark) Potato Dextrose Agar) at 25° C. for 5 days. The floraobtained from the pre-culture were then cut into 3-mm cubic sections,and each section was placed on a petri dish to inoculate 30 seedingswhich had been seeded and grown for 7 days in each dish. The dish wasthen kept at 25° C. After 7 days from plating, green colored survivingplantets were identified as being resisent. The results are shown inFIG. 14. Survival rates on day 7 were 63% for A9 (19 out of 30plantlets) and 0% for the controls (0 out of 30 plantlets),respectively.

[0159] Thus, a transgenic tobacco which expresses a DS9 cDNA in anantisense orientation exhibited resistance to Zhizoctonia solan.

EXAMPLE 14: Paraquat (Tradename) Resistance in Transgenic Plants

[0160] Paraquat is a herbicide which generates superoxide inchloroplasts. A transgenic plant S4 (transgenic tobacco which expressesa DS9 cDNA in a sense orientation, obtained in Example 12) and a wildtype tobacco (control) were grown in a green house. A tobacco leaf wascut into round pieces, and the laef discs were immersed in solutionsrespectively containing 0, 10 and 20 μM paraquat and kept at 25° C.,under light of 5000 lux for 45 hr. The results are shown in FIG. 15. Awild type tobacco showed significant change in color into white afterparaquat treatment at a concectration of 10 μM, while the S4 plantremained green even after the paraquat treatment at a concentration of20 μM, indicating significant suppression of chloroplast degradation.

[0161] Thus, a transgenic tobacco which expresses a DS9 CDNA in a senseorientation exhibited resistance to paraquat.

[0162] According to the present invention, a method for regulating celldeath by regulating an expression level of a cell death regulatory geneis provided. Furthermore, a method for producing a plant which isconferred with resistance to various environmental stress by regulatingcell death is provided. Thus, a plant useful in terms of agriculture andbreeding can be produced. Furthermore, a method for screening aselective inhibitor of a DS9-related gene is provided.

[0163] Various other modifications will be apparent to and can bereadily made by those skilled in the art without departing from thescope and spirit of this invention. Accordingly, it is not intended thatthe scope of the claims appended hereto be limited to the description asset forth herein, but rather that the claims be broadly construed.

1 15 1 2446 DNA Nicotiana tabacum CDS (21)..(2165) DS9 ATP-dependentZn-type metalloprotease 1 aacaccttcc aaaaatagtt atg gcc aat tct ctc ctctct tcc aac ttc atg 53 Met Ala Asn Ser Leu Leu Ser Ser Asn Phe Met 1 510 ggt tct caa atc ttt gtc tct cct ccc acc cct aaa aca aca aag tat 101Gly Ser Gln Ile Phe Val Ser Pro Pro Thr Pro Lys Thr Thr Lys Tyr 15 20 25ttc cat ttt cac tcc aaa aga aag tct tta atc cct caa tca att ctc 149 PheHis Phe His Ser Lys Arg Lys Ser Leu Ile Pro Gln Ser Ile Leu 30 35 40 aacaaa aaa ccc aat tca gat aat tca aag aat att cct tca aaa gct 197 Asn LysLys Pro Asn Ser Asp Asn Ser Lys Asn Ile Pro Ser Lys Ala 45 50 55 gct ttagct gct tta ctc ttt tct tca atc act cca cat gcc tat gct 245 Ala Leu AlaAla Leu Leu Phe Ser Ser Ile Thr Pro His Ala Tyr Ala 60 65 70 75 ctt gataat act acc cct aca gta cca acc cct caa gtg att caa gct 293 Leu Asp AsnThr Thr Pro Thr Val Pro Thr Pro Gln Val Ile Gln Ala 80 85 90 gaa gca gccaat ccc acc act tca aat cca ttc tct caa aat ata atc 341 Glu Ala Ala AsnPro Thr Thr Ser Asn Pro Phe Ser Gln Asn Ile Ile 95 100 105 ttg aat gctcca aag cct caa gca cag acc aat cct gaa ctt cca gaa 389 Leu Asn Ala ProLys Pro Gln Ala Gln Thr Asn Pro Glu Leu Pro Glu 110 115 120 gtt tct caatgg aga tac agt gaa ttc ttg aat gct gtg aaa aag ggt 437 Val Ser Gln TrpArg Tyr Ser Glu Phe Leu Asn Ala Val Lys Lys Gly 125 130 135 aaa gtt gaaagg gtc cga ttc agt aaa gac gga tct gcc ctc ctg ctt 485 Lys Val Glu ArgVal Arg Phe Ser Lys Asp Gly Ser Ala Leu Leu Leu 140 145 150 155 act gctgtt gat ggc cgt aga gct act gta act gtg cct aat gac ccg 533 Thr Ala ValAsp Gly Arg Arg Ala Thr Val Thr Val Pro Asn Asp Pro 160 165 170 gat ttaatt gac att ttg gct atg aat ggt gtt gat ata tca gtt tct 581 Asp Leu IleAsp Ile Leu Ala Met Asn Gly Val Asp Ile Ser Val Ser 175 180 185 gaa ggtgat tct gct ggt aat ggg ttg ttt aat tta att gga aat tta 629 Glu Gly AspSer Ala Gly Asn Gly Leu Phe Asn Leu Ile Gly Asn Leu 190 195 200 ttc cctttt att gct ttt gct gga ttg ttc tat ctt ttc cag aga tct 677 Phe Pro PheIle Ala Phe Ala Gly Leu Phe Tyr Leu Phe Gln Arg Ser 205 210 215 caa ggtggg cct ggt ggg cca ggt ggg ctt ggt ggc ccc atg gat ttt 725 Gln Gly GlyPro Gly Gly Pro Gly Gly Leu Gly Gly Pro Met Asp Phe 220 225 230 235 ggtagg tca aag tca aag ttt caa gaa gtt cct gaa act gga gtg act 773 Gly ArgSer Lys Ser Lys Phe Gln Glu Val Pro Glu Thr Gly Val Thr 240 245 250 tttgct gat gtt gct ggt gct gat caa gct aaa ttg gag tta caa gaa 821 Phe AlaAsp Val Ala Gly Ala Asp Gln Ala Lys Leu Glu Leu Gln Glu 255 260 265 gtggtt gat ttt tta aag aat cct gat aag tat act gct tta ggt gct 869 Val ValAsp Phe Leu Lys Asn Pro Asp Lys Tyr Thr Ala Leu Gly Ala 270 275 280 aaaata cca aaa ggg tgt ctt ttg gtg gga cca cct ggt aca gga aag 917 Lys IlePro Lys Gly Cys Leu Leu Val Gly Pro Pro Gly Thr Gly Lys 285 290 295 acactt ttg gct aga gca gtt gct ggt gaa gct ggt gta cca ttt ttc 965 Thr LeuLeu Ala Arg Ala Val Ala Gly Glu Ala Gly Val Pro Phe Phe 300 305 310 315tca tgt gca gca tca gag ttt gtt gag ttg ttt gtt ggt gtt gga gct 1013 SerCys Ala Ala Ser Glu Phe Val Glu Leu Phe Val Gly Val Gly Ala 320 325 330tct aga gtg agg gat ttg ttc gag aag gcg aag tcg aaa gcg cct tgc 1061 SerArg Val Arg Asp Leu Phe Glu Lys Ala Lys Ser Lys Ala Pro Cys 335 340 345att gtg ttt att gat gag att gat gct gtg ggg agg cag aga ggt gca 1109 IleVal Phe Ile Asp Glu Ile Asp Ala Val Gly Arg Gln Arg Gly Ala 350 355 360gga atg gga ggt ggg aat gat gag aga gag cag act att aat caa ctc 1157 GlyMet Gly Gly Gly Asn Asp Glu Arg Glu Gln Thr Ile Asn Gln Leu 365 370 375ttg act gaa atg gat ggg ttt tct gga aat agt gga gta att gtt ttg 1205 LeuThr Glu Met Asp Gly Phe Ser Gly Asn Ser Gly Val Ile Val Leu 380 385 390395 gct gca acc aat agg cct gat gtt ctt gat tct gca ttg ttg aga cct 1253Ala Ala Thr Asn Arg Pro Asp Val Leu Asp Ser Ala Leu Leu Arg Pro 400 405410 ggg agg ttc gat cga caa gtg act gtc gac agg cct gat gtt gct ggt 1301Gly Arg Phe Asp Arg Gln Val Thr Val Asp Arg Pro Asp Val Ala Gly 415 420425 aga atc aag att ctt cag gtg cat tct aga gga aag gcc ctt gca aag 1349Arg Ile Lys Ile Leu Gln Val His Ser Arg Gly Lys Ala Leu Ala Lys 430 435440 gat gtg gac ttt gag aag att gcc agg aga aca ccg ggt ttc act ggt 1397Asp Val Asp Phe Glu Lys Ile Ala Arg Arg Thr Pro Gly Phe Thr Gly 445 450455 gca gat ttg caa aac ttg atg aat gaa gca gcg atc ctt gca gct agg 1445Ala Asp Leu Gln Asn Leu Met Asn Glu Ala Ala Ile Leu Ala Ala Arg 460 465470 475 cgt gaa cta aag gaa ata agt aaa aat gag ata tct gat gct ttg gag1493 Arg Glu Leu Lys Glu Ile Ser Lys Asn Glu Ile Ser Asp Ala Leu Glu 480485 490 agg ata att gct gga ccg gag aag aaa aat gct gtt gtc tca gag gag1541 Arg Ile Ile Ala Gly Pro Glu Lys Lys Asn Ala Val Val Ser Glu Glu 495500 505 aag aag aag ctg gta gct tat cat gag gcc gcc cat gcc ttg gtt ggt1589 Lys Lys Lys Leu Val Ala Tyr His Glu Ala Ala His Ala Leu Val Gly 510515 520 gca ctt atg ccc gag tat gat cct gtt ccc aag ata tct att att cct1637 Ala Leu Met Pro Glu Tyr Asp Pro Val Pro Lys Ile Ser Ile Ile Pro 525530 535 cgg ggc caa gct ggt ggt ctt acc ttc ttt gcc cct agc gaa gaa aga1685 Arg Gly Gln Ala Gly Gly Leu Thr Phe Phe Ala Pro Ser Glu Glu Arg 540545 550 555 ctt gag tcg ggc ttg tac agc agg agc tac cta gag aat caa atggca 1733 Leu Glu Ser Gly Leu Tyr Ser Arg Ser Tyr Leu Glu Asn Gln Met Ala560 565 570 gtt gca ctt ggt gga agg gtt gct gag gag gtt att ttt gga caagac 1781 Val Ala Leu Gly Gly Arg Val Ala Glu Glu Val Ile Phe Gly Gln Asp575 580 585 aac gta aca act ggg gca tct aac gat ttc atg ctt gtt tca cgagtg 1829 Asn Val Thr Thr Gly Ala Ser Asn Asp Phe Met Leu Val Ser Arg Val590 595 600 gca agg cag atg gtt gag aga tta ggg ttc acc aca aag atc ggacag 1877 Ala Arg Gln Met Val Glu Arg Leu Gly Phe Thr Thr Lys Ile Gly Gln605 610 615 gtt gcc att gga gga ggt gga gga aat cct ttc cta ggt caa cagatg 1925 Val Ala Ile Gly Gly Gly Gly Gly Asn Pro Phe Leu Gly Gln Gln Met620 625 630 635 tca acc cag aaa gac tac tcc atg gca aca gcc gat gtg gttgat gct 1973 Ser Thr Gln Lys Asp Tyr Ser Met Ala Thr Ala Asp Val Val AspAla 640 645 650 gaa gta agg gaa ttg gtt gaa aga gca tat gaa agg gca acacag att 2021 Glu Val Arg Glu Leu Val Glu Arg Ala Tyr Glu Arg Ala Thr GlnIle 655 660 665 atc aca aca cac att gac atc cta cac aag ctt gct cag ctgttg ata 2069 Ile Thr Thr His Ile Asp Ile Leu His Lys Leu Ala Gln Leu LeuIle 670 675 680 gag aaa gaa act gtt gat ggt gaa gag ttc atg agc ctt ttcatc gat 2117 Glu Lys Glu Thr Val Asp Gly Glu Glu Phe Met Ser Leu Phe IleAsp 685 690 695 ggc aag gcc gag cta tac att tct tgg gtc tct aag gag gaggat 2162 Gly Lys Ala Glu Leu Tyr Ile Ser Trp Val Ser Lys Glu Glu Asp 700705 710 tagtttctgg cttaacaaga cttgatgtat ctggtggttg agagtggtaaattgctgatg 2222 caagagaaat aggcataata catagtgctt tagactgaag aaattgcattgcagaaccaa 2282 cattttcttc cataagtttg gccacttgcc tttctgtacc atcacttgaccacttttccc 2342 aggctggttg gttatttcca acttcactgc tctcttccta aataagacaagccacaaaaa 2402 gggataaatt attaattgat aggttggaca attctgcaaa aaaa 2446 2714 PRT Nicotiana tabacum 2 Met Ala Asn Ser Leu Leu Ser Ser Asn Phe MetGly Ser Gln Ile Phe 1 5 10 15 Val Ser Pro Pro Thr Pro Lys Thr Thr LysTyr Phe His Phe His Ser 20 25 30 Lys Arg Lys Ser Leu Ile Pro Gln Ser IleLeu Asn Lys Lys Pro Asn 35 40 45 Ser Asp Asn Ser Lys Asn Ile Pro Ser LysAla Ala Leu Ala Ala Leu 50 55 60 Leu Phe Ser Ser Ile Thr Pro His Ala TyrAla Leu Asp Asn Thr Thr 65 70 75 80 Pro Thr Val Pro Thr Pro Gln Val IleGln Ala Glu Ala Ala Asn Pro 85 90 95 Thr Thr Ser Asn Pro Phe Ser Gln AsnIle Ile Leu Asn Ala Pro Lys 100 105 110 Pro Gln Ala Gln Thr Asn Pro GluLeu Pro Glu Val Ser Gln Trp Arg 115 120 125 Tyr Ser Glu Phe Leu Asn AlaVal Lys Lys Gly Lys Val Glu Arg Val 130 135 140 Arg Phe Ser Lys Asp GlySer Ala Leu Leu Leu Thr Ala Val Asp Gly 145 150 155 160 Arg Arg Ala ThrVal Thr Val Pro Asn Asp Pro Asp Leu Ile Asp Ile 165 170 175 Leu Ala MetAsn Gly Val Asp Ile Ser Val Ser Glu Gly Asp Ser Ala 180 185 190 Gly AsnGly Leu Phe Asn Leu Ile Gly Asn Leu Phe Pro Phe Ile Ala 195 200 205 PheAla Gly Leu Phe Tyr Leu Phe Gln Arg Ser Gln Gly Gly Pro Gly 210 215 220Gly Pro Gly Gly Leu Gly Gly Pro Met Asp Phe Gly Arg Ser Lys Ser 225 230235 240 Lys Phe Gln Glu Val Pro Glu Thr Gly Val Thr Phe Ala Asp Val Ala245 250 255 Gly Ala Asp Gln Ala Lys Leu Glu Leu Gln Glu Val Val Asp PheLeu 260 265 270 Lys Asn Pro Asp Lys Tyr Thr Ala Leu Gly Ala Lys Ile ProLys Gly 275 280 285 Cys Leu Leu Val Gly Pro Pro Gly Thr Gly Lys Thr LeuLeu Ala Arg 290 295 300 Ala Val Ala Gly Glu Ala Gly Val Pro Phe Phe SerCys Ala Ala Ser 305 310 315 320 Glu Phe Val Glu Leu Phe Val Gly Val GlyAla Ser Arg Val Arg Asp 325 330 335 Leu Phe Glu Lys Ala Lys Ser Lys AlaPro Cys Ile Val Phe Ile Asp 340 345 350 Glu Ile Asp Ala Val Gly Arg GlnArg Gly Ala Gly Met Gly Gly Gly 355 360 365 Asn Asp Glu Arg Glu Gln ThrIle Asn Gln Leu Leu Thr Glu Met Asp 370 375 380 Gly Phe Ser Gly Asn SerGly Val Ile Val Leu Ala Ala Thr Asn Arg 385 390 395 400 Pro Asp Val LeuAsp Ser Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg 405 410 415 Gln Val ThrVal Asp Arg Pro Asp Val Ala Gly Arg Ile Lys Ile Leu 420 425 430 Gln ValHis Ser Arg Gly Lys Ala Leu Ala Lys Asp Val Asp Phe Glu 435 440 445 LysIle Ala Arg Arg Thr Pro Gly Phe Thr Gly Ala Asp Leu Gln Asn 450 455 460Leu Met Asn Glu Ala Ala Ile Leu Ala Ala Arg Arg Glu Leu Lys Glu 465 470475 480 Ile Ser Lys Asn Glu Ile Ser Asp Ala Leu Glu Arg Ile Ile Ala Gly485 490 495 Pro Glu Lys Lys Asn Ala Val Val Ser Glu Glu Lys Lys Lys LeuVal 500 505 510 Ala Tyr His Glu Ala Ala His Ala Leu Val Gly Ala Leu MetPro Glu 515 520 525 Tyr Asp Pro Val Pro Lys Ile Ser Ile Ile Pro Arg GlyGln Ala Gly 530 535 540 Gly Leu Thr Phe Phe Ala Pro Ser Glu Glu Arg LeuGlu Ser Gly Leu 545 550 555 560 Tyr Ser Arg Ser Tyr Leu Glu Asn Gln MetAla Val Ala Leu Gly Gly 565 570 575 Arg Val Ala Glu Glu Val Ile Phe GlyGln Asp Asn Val Thr Thr Gly 580 585 590 Ala Ser Asn Asp Phe Met Leu ValSer Arg Val Ala Arg Gln Met Val 595 600 605 Glu Arg Leu Gly Phe Thr ThrLys Ile Gly Gln Val Ala Ile Gly Gly 610 615 620 Gly Gly Gly Asn Pro PheLeu Gly Gln Gln Met Ser Thr Gln Lys Asp 625 630 635 640 Tyr Ser Met AlaThr Ala Asp Val Val Asp Ala Glu Val Arg Glu Leu 645 650 655 Val Glu ArgAla Tyr Glu Arg Ala Thr Gln Ile Ile Thr Thr His Ile 660 665 670 Asp IleLeu His Lys Leu Ala Gln Leu Leu Ile Glu Lys Glu Thr Val 675 680 685 AspGly Glu Glu Phe Met Ser Leu Phe Ile Asp Gly Lys Ala Glu Leu 690 695 700Tyr Ile Ser Trp Val Ser Lys Glu Glu Asp 705 710 3 300 PRT Nicotianatabacum DS9 (positions 273-572) 3 Lys Asn Pro Asp Lys Tyr Thr Ala LeuGly Ala Lys Ile Pro Lys Gly 1 5 10 15 Cys Leu Leu Val Gly Pro Pro GlyThr Gly Lys Thr Leu Leu Ala Arg 20 25 30 Ala Val Ala Gly Glu Ala Gly ValPro Phe Phe Ser Cys Ala Ala Ser 35 40 45 Glu Phe Val Glu Leu Phe Val GlyVal Gly Ala Ser Arg Val Arg Asp 50 55 60 Leu Phe Glu Lys Ala Lys Ser LysAla Pro Cys Ile Val Phe Ile Asp 65 70 75 80 Glu Ile Asp Ala Val Gly ArgGln Arg Gly Ala Gly Met Gly Gly Gly 85 90 95 Asn Asp Glu Arg Glu Gln ThrIle Asn Gln Leu Leu Thr Glu Met Asp 100 105 110 Gly Phe Ser Gly Asn SerGly Val Ile Val Leu Ala Ala Thr Asn Arg 115 120 125 Pro Asp Val Leu AspSer Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg 130 135 140 Gln Val Thr ValAsp Arg Pro Asp Val Ala Gly Arg Ile Lys Ile Leu 145 150 155 160 Gln ValHis Ser Arg Gly Lys Ala Leu Ala Lys Asp Val Asp Phe Glu 165 170 175 LysIle Ala Arg Arg Thr Pro Gly Phe Thr Gly Ala Asp Leu Gln Asn 180 185 190Leu Met Asn Glu Ala Ala Ile Leu Ala Ala Arg Arg Glu Leu Lys Glu 195 200205 Ile Ser Lys Asn Glu Ile Ser Asp Ala Leu Glu Arg Ile Ile Ala Gly 210215 220 Pro Glu Lys Lys Asn Ala Val Val Ser Glu Glu Lys Lys Lys Leu Val225 230 235 240 Ala Tyr His Glu Ala Ala His Ala Leu Val Gly Ala Leu MetPro Glu 245 250 255 Tyr Asp Pro Val Pro Lys Ile Ser Ile Ile Pro Arg GlyGln Ala Gly 260 265 270 Gly Leu Thr Phe Phe Ala Pro Ser Glu Glu Arg LeuGlu Ser Gly Leu 275 280 285 Tyr Ser Arg Ser Tyr Leu Glu Asn Gln Met AlaVal 290 295 300 4 300 PRT Arabidopsis thaliana ArFtsH (positions282-581) 4 Lys Asn Pro Asp Lys Tyr Thr Ala Leu Gly Ala Lys Ile Pro LysGly 1 5 10 15 Cys Leu Leu Val Gly Pro Pro Gly Thr Gly Lys Thr Leu LeuAla Arg 20 25 30 Ala Val Ala Gly Glu Ala Gly Val Pro Phe Phe Ser Ser ArgPro Gln 35 40 45 Glu Phe Val Glu Leu Phe Val Gly Val Gly Ala Ser Arg ValArg Asp 50 55 60 Leu Phe Glu Lys Ala Lys Ser Lys Ala Pro Cys Ile Val PheIle Asp 65 70 75 80 Glu Ile Asp Ala Val Gly Arg Gln Arg Gly Ala Gly MetGly Gly Gly 85 90 95 Asn Asp Glu Arg Glu Gln Thr Ile Asn Gln Leu Leu ThrGlu Met Asp 100 105 110 Gly Phe Ser Gly Asn Ser Gly Val Ile Val Leu AlaAla Thr Asn Arg 115 120 125 Pro Asp Val Leu Asp Ser Ala Leu Leu Arg ProGly Arg Phe Asp Arg 130 135 140 Gln Val Thr Val Asp Arg Pro Asp Val AlaGly Arg Val Lys Ile Leu 145 150 155 160 Gln Val His Ser Arg Gly Lys AlaLeu Gly Lys Asp Val Asp Phe Asp 165 170 175 Lys Val Ala Arg Arg Thr ProGly Phe Thr Gly Ala Asp Leu Gln Asn 180 185 190 Leu Met Asn Glu Ala AlaIle Leu Ala Ala Arg Arg Asp Val Lys Glu 195 200 205 Ile Ser Lys Asp GluIle Ser Asp Ala Leu Glu Arg Ile Ile Ala Gly 210 215 220 Pro Glu Lys LysAsn Ala Val Val Ser Glu Glu Lys Lys Arg Leu Val 225 230 235 240 Ala TyrHis Glu Ala Gly His Ala Leu Val Gly Ala Leu Met Pro Glu 245 250 255 TyrAsp Pro Val Ala Lys Ile Ser Ile Ile Pro Arg Gly Gln Ala Gly 260 265 270Gly Leu Thr Phe Phe Ala Pro Ser Glu Glu Arg Leu Glu Ser Gly Leu 275 280285 Tyr Ser Arg Ser Tyr Leu Glu Asn Gln Met Ala Val 290 295 300 5 297PRT Escherichia coli FtsH (positions 172-468) 5 Arg Glu Pro Ser Arg PheGln Lys Leu Gly Gly Lys Ile Pro Lys Gly 1 5 10 15 Val Leu Met Val GlyPro Pro Gly Thr Gly Lys Thr Leu Leu Ala Lys 20 25 30 Ala Ile Ala Gly GluAla Lys Val Pro Phe Phe Thr Ile Ser Gly Ser 35 40 45 Asp Phe Val Glu MetPhe Val Gly Val Gly Ala Ser Arg Val Arg Asp 50 55 60 Met Phe Glu Gln AlaLys Lys Ala Ala Pro Cys Ile Ile Phe Ile Asp 65 70 75 80 Glu Ile Asp AlaVal Gly Arg Gln Arg Gly Ala Gly Leu Gly Gly Gly 85 90 95 His Asp Glu ArgGlu Gln Thr Ile Asn Gln Met Leu Val Glu Met Asp 100 105 110 Gly Phe GluGly Asn Glu Gly Ile Ile Val Ile Ala Ala Thr Asn Arg 115 120 125 Pro AspVal Leu Asp Pro Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg 130 135 140 GlnVal Val Val Gly Leu Pro Asp Val Arg Gly Arg Glu Gln Ile Leu 145 150 155160 Lys Val His Met Arg Arg Val Pro Leu Ala Pro Asp Ile Asp Ala Ala 165170 175 Ile Ile Ala Arg Gly Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala Asn180 185 190 Leu Val Asn Glu Ala Ala Leu Phe Ala Ala Arg Gly Asn Lys ArgVal 195 200 205 Val Ser Met Val Glu Phe Glu Lys Ala Lys Asp Lys Ile MetMet Gly 210 215 220 Ala Glu Arg Arg Ser Met Val Met Thr Glu Ala Gln LysGlu Ser Thr 225 230 235 240 Ala Tyr His Glu Ala Gly His Ala Ile Ile GlyArg Leu Val Pro Glu 245 250 255 His Asp Pro Val His Lys Val Thr Ile IlePro Arg Gly Arg Ala Leu 260 265 270 Gly Val Thr Phe Phe Leu Pro Glu GlyAsp Ala Ile Ser Ala Ser Arg 275 280 285 Gln Lys Leu Glu Ser Gln Ile SerThr 290 295 6 294 PRT Saccharomyces cerevisiae yeast Osd1p (positions301-594) 6 Lys Asp Pro Thr Lys Tyr Glu Ser Leu Gly Gly Lys Leu Pro LysGly 1 5 10 15 Val Leu Leu Thr Gly Pro Pro Gly Thr Gly Lys Thr Leu LeuAla Arg 20 25 30 Ala Thr Ala Gly Glu Ala Gly Val Asp Phe Phe Phe Met SerGly Ser 35 40 45 Glu Phe Asp Glu Val Tyr Val Gly Val Gly Ala Lys Arg IleArg Asp 50 55 60 Leu Phe Ala Gln Ala Arg Ser Arg Ala Pro Ala Ile Ile PheIle Asp 65 70 75 80 Glu Leu Asp Ala Ile Gly Gly Lys Arg Asn Pro Lys AspGln Ala Tyr 85 90 95 Ala Lys Gln Thr Leu Asn Gln Leu Leu Val Glu Leu AspGly Phe Ser 100 105 110 Gln Thr Ser Gly Ile Ile Ile Ile Gly Ala Thr AsnPhe Pro Glu Ala 115 120 125 Leu Asp Lys Ala Leu Leu Arg Pro Gly Arg PheAsp Lys Val Val Asn 130 135 140 Val Asp Leu Pro Asp Val Arg Gly Arg AlaAsp Ile Leu Lys His His 145 150 155 160 Met Lys Lys Ile Thr Leu Ala AspAsn Val Asp Pro Thr Ile Ile Ala 165 170 175 Arg Gly Thr Pro Gly Leu SerGly Ala Glu Leu Ala Asn Leu Val Asn 180 185 190 Gln Ala Ala Val Tyr AlaCys Gln Lys Asn Ala Val Ser Val Asp Met 195 200 205 Ser His Phe Glu TrpAla Lys Asp Lys Ile Leu Met Gly Ala Glu Arg 210 215 220 Lys Thr Met ValLeu Thr Asp Ala Ala Arg Lys Ala Thr Ala Phe His 225 230 235 240 Glu AlaGly His Ala Ile Met Ala Lys Tyr Thr Asn Gly Ala Thr Pro 245 250 255 LeuTyr Lys Ala Thr Ile Leu Pro Arg Gly Arg Ala Leu Gly Ile Thr 260 265 270Phe Gln Leu Pro Glu Met Asp Lys Val Asp Ile Thr Lys Arg Glu Cys 275 280285 Gln Ala Arg Leu Asp Val 290 7 296 PRT Capsicum sp. red pepper Pftf(positions 250-545) 7 Lys Lys Pro Glu Arg Phe Thr Ala Val Gly Ala ArgIle Pro Lys Gly 1 5 10 15 Val Leu Leu Val Gly Pro Pro Gly Thr Gly LysThr Leu Leu Ala Lys 20 25 30 Ala Ile Ala Gly Glu Ala Gly Val Pro Phe PheSer Ile Ser Gly Ser 35 40 45 Glu Phe Val Glu Met Phe Val Gly Val Gly AlaSer Arg Val Arg Asp 50 55 60 Leu Phe Lys Lys Ala Lys Glu Asn Ala Pro CysIle Val Phe Val Asp 65 70 75 80 Glu Ile Asp Ala Val Gly Arg Gln Arg GlyThr Gly Ile Gly Gly Gly 85 90 95 Asn Asp Glu Arg Glu Gln Thr Leu Asn GlnLeu Leu Thr Glu Met Asp 100 105 110 Gly Phe Glu Gly Asn Thr Gly Ile IleVal Val Ala Ala Thr Asn Arg 115 120 125 Ala Asp Ile Leu Asp Ser Ala LeuLeu Arg Pro Gly Arg Phe Asp Arg 130 135 140 Gln Val Ser Val Asp Val ProAsp Ile Lys Gly Arg Thr Glu Ile Leu 145 150 155 160 Lys Val His Ala GlyAsn Lys Lys Phe Asp Ser Asp Val Ser Leu Glu 165 170 175 Val Ile Ala MetArg Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala Asn 180 185 190 Leu Leu AsnGlu Ala Ala Ile Leu Ala Gly Arg Arg Gly Lys Thr Ala 195 200 205 Ile AlaSer Lys Glu Ile Asp Asp Ser Ile Asp Arg Ile Val Ala Gly 210 215 220 MetGlu Gly Thr Val Met Thr Asp Gly Lys Ser Lys Ser Leu Val Ala 225 230 235240 Tyr His Glu Val Gly His Ala Ile Cys Gly Thr Leu Thr Pro Gly His 245250 255 Asp Pro Val Gln Lys Val Thr Leu Ile Pro Arg Gly Gln Ala Lys Gly260 265 270 Leu Thr Trp Phe Ile Pro Ala Asp Asp Pro Thr Leu Ile Ser LysGln 275 280 285 Gln Leu Phe Ala Arg Ile Val Gly 290 295 8 32 DNAArtificial Sequence Description of Artificial SequencePCR primer forGST-DS9 fusion gene 8 acgtggatcc ttgaatgctg tgaaaaaggg ta 32 9 32 DNAArtificial Sequence Description of Artificial SequencePCR primer forGST-DS9 fusion gene 9 acgtgaattc ttatgcctat ttctcttgca tc 32 10 24 DNAArtificial Sequence Description of Artificial SequencePCR primer A foracidic PR-1 protein cDNA 10 tactaattga aacgacctac gtcc 24 11 27 DNAArtificial Sequence Description of Artificial SequencePCR primer B foracidic PR-1 protein cDNA 11 ataataatat ctgatcatac atcaagc 27 12 22 DNAArtificial Sequence Description of Artificial SequencePCR primer A forbasic PR-1 protein cDNA 12 atccctttga ttccaaggtt gg 22 13 26 DNAArtificial Sequence Description of Artificial SequencePCR primer B forbasic PR-1 protein cDNA 13 caaaacacat acatatacac acctcc 26 14 20 DNAArtificial Sequence Description of Artificial SequencePCR primer A forDS9 coding region 14 actatggcca attctctctc 20 15 22 DNA ArtificialSequence Description of Artificial SequencePCR primer B for DS9 codingregion 15 ttatgcctat ttctcttgca tc 22

What is claimed is:
 1. A method for regulating cell death in a plant,comprising the steps of: transforming a plant cell with a polynucleotidecontaining a gene encoding DS9 or a homologue thereof or a part of thegene; and redifferentiating the transformed plant cell to obtain aplant, wherein the DS9 or the homologue thereof is an ATP-dependentZn-type metalloprotease, and the polynucleotide decreases or increasesproduction of the ATP-dependent Zn-type metalloprotease in the plantcell, whereby cell death of a cell in the plant is promoted orsuppressed.
 2. A method according to claim 1, wherein the polynucleotidecontains the gene encoding the DS9 or the homologue thereof or the partof the gene in an antisense orientation, whereby cell death of a cell inthe plant is promoted.
 3. A method for producing a plant which isconferred with resistance to environmental stress, comprising the stepsof: transforming a plant cell with a polynucleotide containing a geneencoding DS9 or a homologue thereof or a part of the gene; andredifferentiating the transformed plant cell to obtain a plant, whereinthe DS9 or the homologue thereof is an ATP-dependent Zn-typemetalloprotease, and the polynucleotide decreases or increasesproduction of the ATP-dependent Zn-type metalloprotease in the plantcell.
 4. A method according to claim 3, wherein the environmental stressis pathogen infection.
 5. A method according to claim 3, wherein thenucleotide contains the gene encoding the DS9 or the homologue thereofor the part of the gene in an antisense orientation.
 6. A methodaccording to claim 3, wherein the homologue has a homology of about 70%or more with respect to an ATPase region of the DS9.
 7. A method forscreening a selective inhibitor of a gene encoding DS9 or a homologuethereof, wherein the DS9 or the homologue thereof is an ATP-dependentZn-type metalloprotease, comprising the steps of: introducing acandidate inhibitor into a plant cell having a gene encoding the DS9 orthe homologue thereof; and identifying whether or not production of theDS9 or the homologue thereof is selectively decreased in the plant cell.